Myosin II plays fundamental roles in cytokinesis, cell migration, and cell shape changes during development. In all these settings, it is well established that dynamic localized assembly of myosin into the cytoskeleton is critical for its cellular contractile roles. Despite the importance of spatially and temporally regulated assembly, the signaling mechanisms that control localized assembly and disassembly of myosin II are not understood in any system. We are using the simple amoeba Dictyostelium discoideum as a model system for identifying signaling pathways that regulate myosin assembly. This simple amoeba displays forms of cellular motility, chemotaxis, and second messenger signaling similar to those displayed by motile mammalian cells such as neutrophils or macrophages. Myosin II assembly in this system is regulated by phosphorylation/dephosphorylation of a set of mapped threonine residues that lie near the tip of the myosin tail. We have previously identified a set of novel myosin heavy chain kinases (MHCKs) in this system, that participate in the in vivo control of myosin assembly via phosphorylation of the mapped target sites in the myosin tail. These enzymes are now recognized as the prototype for a highly novel family of protein kinases present in Dictyostelium and throughout the animal kingdom known as "Alpha kinases". Recently, 1 mammalian alpha kinase (TRPM7) has been implicated in apoptotic pathway activation in response to oxidative stress, but roles of other mammalian alpha kinases are largely unknown. Our molecular and genomic approaches in Dictyostelium have revealed a total of 6 alpha kinases in this simple model organism. At least 2 of the Dictyostelium alpha kinases appear NOT to be MHC kinases, but likely serve other roles in the cell. We propose a series of complementary approaches to gain insights into the roles of all of these enzymes. In this proposal we will: (1) use biochemistry/cellular analysis to elucidate the role of autophosphorylation in MHCK activation, (2) perform studies to test the cellular role of an MHC phosphatase that we identified biochemically in earlier work, (3) perform further cellular/biochemical analysis of VwkA and AK1, 2 remaining Dictyostelium alpha kinases for which cellular roles are not well understood, and (4) perform genetic screens to identify new genes involved in myosin II-related cellular functions in settings ranging from cell division to osmotic protective responses. Our studies will provide an important foundation of direct relevance for understanding myosin II regulation in mammalian systems. The studies we propose addressing the full complement of Dictyostelium alpha kinases will also provide a powerful platform for comparisons and insights into possible roles of alpha kinases in mammalian systems.